Compositions and methods for diagnosing and monitoring hyperthyroidism in a feline

ABSTRACT

The present invention provides a method of diagnosing the existence or risk of hyperthyroidism in a feline comprising measuring the level of expression of one or more biomarkers selected from the group consisting of, e.g., IYD, TG, SLC5A5, NIS, TPO, TSHR, DUOX1, DUOX2 (ThOX), TGFB1, CSTD, DCN and SEPP1 and the expression products thereof, in a biological sample from the feline, wherein elevated expression of the one or more biomarkers in the sample relative to a control value for expression in a sample from a normal feline or feline population, or a baseline value from the feline, indicates the existence or risk of hyperthyroidism; a method of treating a feline so diagnosed; and compositions, reagents and kits for carrying out the specified methods.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/125,981, filed on Nov. 25, 2014, which is a national stageentry under 35 U.S.C. § 371 of International Patent Application No.PCT/US2012/042534, filed 14 Jun. 2012, which claims priority to U.S.Provisional Patent Application Ser. No. 61/497,264, filed on 15 Jun.2011, both of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions, materials and methods fordiagnosing and/or monitoring hyperthyroidism in felines and diseases anddisorders in felines relating to or resulting from hyperthyroidism.

BACKGROUND OF THE INVENTION

Hyperthyroidism is the most common hormone abnormality in felines. It ismost common in older felines, and somewhat more common in females thanin males. Hyperthyroidism is caused by overproduction of thyroidhormones, particularly thyroxine or T-4. Overproduction of T-4 candramatically increase the animal's basal metabolic rate, leading toweight loss, increased appetite, restlessness, poor hair coat,tachycardia, increased drinking and urination, vomiting, diarrhea,panting, difficulty breathing, fever, elevated blood pressure, andeventually weakness, listlessness, muscle tremors, wasting, and death.Advanced hyperthyroidism is often associated with kidney problems andheart problems. Effective treatments include use of drugs such asmethimazole or carbimazole, which inhibit production of thyroidhormones, radioiodine therapy, which destroys overactive thyroid cells,and surgical thyroidectomy. If the disease is detected too late,however, the damage may be irreversible. Accordingly, early diagnosis iscritical.

The most common test for diagnosing hyperthyroidism is a blood test forT-4, where significantly elevated T-4 levels indicate hyperthyroidism,or a T-3 suppression test, which measures suppression of T-4 in responseto administration of T-3, wherein absence of suppression indicate ahyperthyroid condition. However, a feline's level of T-3 and T-4 mayfluctuate in the course of the day, rendering tests unreliable, andthere are also various diseases that can artificially lower T-4 levels,thus masking a hyperthyroid condition. Radioimaging of the thyroid usingtechnetium is possible, and may be useful to detect tumors or abnormalthyroid tissues, but it is expensive.

Accordingly, there is a need for alternative efficient and effectivemethods of detecting hyperthyroidism in felines.

A number of methods have been developed for studying differential geneexpression, e.g., DNA microarrays, expressed tag sequencing (EST),serial analysis of gene expression (SAGE), subtractive hybridization,subtractive cloning and differential display (DD) for mRNA,RNA-arbitrarily primed PCR (RAP-PCR), real-time PCR (RT-PCR),representational difference analysis (RDA), two-dimensional gelelectrophoresis, mass spectrometry, and protein microarray basedantibody-binding for proteins.

Due to the complexity of the biological pathways implicated inhyperthyroidism and the inherent molecular interactions andintercellular signaling processes, it is highly desirable to understandat a genetic level the interactions that are taking place. Detection ofdysregulated genes in the early stages of hyperthyroidism in felines ishelpful in understanding the biology of hyperthyroidism in felines on agenome-wide basis, which would be helpful in designing methods fordetermining the risk of developing, predisposition for, diagnosing of,and devising and monitoring a treatment plan for hyperthyroidism.

A more detailed understanding of the biological pathways involvedthrough gene expression profiling would aid in the development ofdiagnostic procedures, reagents and test kits as well as salutarypharmaceutical, nutraceutical and nutritional (dietary) interventions inthe disease pathways. These approaches may enable early detection andpotentially prevention or treatment of the underlying disorder.Dysregulated genes involved in the pathology of thyroid disorders mayserve as important biomarkers for diagnosis and potentially preventionor treatment of the disorder and to optimize selection of appropriatepharmaceutical, nutraceutical and nutritional (dietary) interventions.

The level of gene expression and/or the determination of the level offunctioning of an expressed gene product in a feline may be used toselect an appropriate agent for therapeutic or prophylactic use. Thisdata may be employed by the skilled worker in selecting appropriatedrugs as agents for the prevention or treatment of renal diseases infelines through gene expression profiling. Gene expression data andanalysis may also be used to select nutritional compositions, dietarysupplements, and nutraceuticals having a salutary effect on promotingnormal thyroid function performance by utilizing biomarkers indicativeof a healthy state of kidney functioning.

Only very limited work has been done to date in screening the felinegenome for gene expression profiles in connection with the diagnosis ofdiseases in felines. Studies in healthy populations of felines versuspopulations having a disease such as hyperthyroidism as described inthis specification have not been extensively conducted. Little data isavailable with respect to the expression profile of the feline genome,especially with respect to the development of renal diseases in felinesover time.

Hyperthyroidism is a leading cause of death in felines. To effectivelytreat hyperthyroidism, early diagnosis and treatment is essential,before there is irreversible damage to the heart and/or kidneys.Accordingly, there is a need for better methods to identify animalshaving hyperthyroidism or at elevated risk for hyperthyroidism, so thatthey can be treated appropriately.

SUMMARY OF THE INVENTION

The inventors have studied gene expression in thyroid tissue and wholeblood in felines with hyperthyroidism. In the tissue study, 1308significant differential expression genes are found between hyperthyroidfelines and non-hyperthyroid felines with FDR (False discovery rate)=0.1and fold change of >=+/−1.25 in expression. In the whole blood study,1094 significant genes are found between hyperthyroid felines andnon-hyperthyroid felines with the same cutoff as shown above. In thetissue study, all the genes known to be involved in the pathway leadingto the production of thyroid hormones are found to be up-regulated inthe hyperthyroid felines. The increased expression of these genesclearly contributes to a high T4 production in the feline body. In thewhole blood study, 60 genes are found to overlap with those found fromthe solid tissue work that appear to be regulated in the same directionas seen in the tissue study. By using these 60 genes, the hyperthyroidfelines can be distinguished from the normal felines as tested in thewhole blood, as well as in the tissue. These 60 genes, listed in TablesA and B below, may permit detection of feline hyperthyroidism.

Accordingly, the present invention provides compositions, materials andmethods for diagnosing and/or monitoring hyperthyroidism in felines anddiseases and disorders in felines relating to or resulting fromhyperthyroidism, including compositions and methods for: determining therisk of developing, identifying a predisposition for, diagnosing of,devising and monitoring a treatment plan for, and monitoring the statusof hyperthyroidism in a feline, wherein the hyperthyroidism isdetectable by utilizing at least one relevant biomarker isolated andmeasured from a biological test sample taken from such feline, whereinthe expression of the biomarker correlates positively or negatively withthe disease. A relevant biomarker for practice of the compositions andmethods of the present invention comprises a polynucleotide or proteinpresent in such biological test sample of such feline. A biological testsample for the practice of the method of the invention may comprise, forexample, a tissue sample from such feline. The biomarkers are selectedalso on the basis of being secreted, so they can be detected in bloodserum or plasma, or in urine. Accordingly the biological test sample mayalso be a specimen of a biological fluid taken from such feline, forexample blood or urine.

Genes which are found to be particularly up-regulated in thyroid hormoneover-production include:

IYD (4.55 fold increase): This gene encodes an enzyme that catalyzes theoxidative NADPH-dependent deiodination of mono- and diiodotyrosine,which are the halogenated byproducts of thyroid hormone production. TheN-terminus of the protein functions as a membrane anchor. Mutations inthis gene cause congenital hypothyroidism due to dyshormonogenesis type4, which is also referred to as deiodinase deficiency, or iodotyrosinedehalogenase deficiency, or thyroid hormonogenesis type 4.

TG (2.02 fold increase): Thyroglobulin (Tg) is a glycoprotein homodimerproduced predominantly by the thyroid gland. It acts as a substrate forthe synthesis of thyroxine and triiodothyronine as well as the storageof the inactive forms of thyroid hormone and iodine. Thyroglobulin issecreted from the endoplasmic reticulum to its site of iodination, andsubsequent thyroxine biosynthesis, in the follicular lumen. Mutations inthis gene cause thyroid dyshormonogenesis, manifested as goiter, and areassociated with moderate to severe congenital hypothyroidism.

SLC5A5, NIS (4.6 fold increase): This gene encodes a member of thesodium glucose cotransporter family. The encoded protein is responsiblefor the uptake of iodine in tissues such as the thyroid and lactatingbreast tissue. The iodine taken up by the thyroid is incorporated intothe metabolic regulators triiodothyronine (T3) and tetraiodothyronine(T4). Mutations in this gene are associated with thyroiddyshormonogenesis 1.

TPO (4.06 fold increase): This gene encodes a membrane-boundglycoprotein. The encoded protein acts as an enzyme and plays a centralrole in thyroid gland function. The protein functions in the iodinationof tyrosine residues in thyroglobulin and phenoxyester formation betweenpairs of iodinated tyrosines to generate the thyroid hormones, thyroxineand triiodothyronine. Mutations in this gene are associated with severaldisorders of thyroid hormonogenesis, including congenitalhypothyroidism, congenital goiter, and thyroid hormone organificationdefect IIA.

TSHR (2.2 fold increase): The protein encoded by this gene is a membraneprotein and a major controller of thyroid cell metabolism. The encodedprotein is a receptor for thyrothropin and thyrostimulin, and itsactivity is mediated by adenylate cyclase. Defects in this gene are acause of several types of hyperthyroidism.

DUOX2, ThOX (1.55 fold increase): The protein encoded by this gene is aglycoprotein and a member of the NADPH oxidase family. The synthesis ofthyroid hormone is catalyzed by a protein complex located at the apicalmembrane of thyroid follicular cells. This complex contains an iodidetransporter, thyroperoxidase, and a peroxide generating system thatincludes this encoded protein and DUOX1. This protein is known as dualoxidase because it has both a peroxidase homology domain and a gp91phoxdomain.

Other genes which are up-regulated in both blood and thyroid tissue ofhyperthyroid felines include the following:

TABLE A CHI3L1 chitinase 3-like 1 (cartilage glycoprotein-39) PDZK1IP1PDZK1 interacting protein 1 MAPK13 mitogen-activated protein kinase 13PGD phosphogluconate dehydrogenase MBOAT7 membrane boundO-acyltransferase domain containing 7 CTSD felinehepsin D CYP4F3cytochrome P450, family 4, subfamily F, polypeptide 3 TGFB1 transforminggrowth factor, beta 1 C20orf3 chromosome 20 open reading frame 3 ACAA2acetyl-CoA acyltransferase 2 HTATIP2 HIV-1 Tat interactive protein 2, 30kDa RHOC ras homolog gene family, member C G6PD glucose-6-phosphatedehydrogenase GSTP1 glutathione S-transferase pi 1 CAPG capping protein(actin filament), gelsolin-like AP1S1 adaptor-related protein complex 1,sigma 1 subunit ATP6V1D ATPase, H+ transporting, lysosomal 34 kDa, V1subunit D CAPN1 calpain 1, (mu/I) large subunit LASS4 LAG1 homolog,ceramide synthase 4 PDCL phosducin-like HSD3B7 hydroxy-delta-5-steroiddehydrogenase, 3 beta- and steroid delta-isomerase 7 LRPAP1 low densitylipoprotein receptor-related protein associated protein 1 PARP3 poly(ADP-ribose) polymerase family, member 3 ATP6AP1 ATPase, H+transporting, lysosomal accessory protein 1 B3GNT8 UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyl- transferase 8 SORT1 sortilin 1

Thus, for example, up-regulation of the genes on this table A can serveas biomarkers for hyperthyroidism. These genes are not previously knownto be involved in hyperthyroidism. For example, TGFB1 encodes a memberof the transforming growth factor beta (TGFB) family of cytokines, whichare multifunctional peptides that regulate proliferation,differentiation, adhesion, migration, and other functions in many celltypes. The protein positively and negatively regulates many other growthfactors. The secreted protein is cleaved into a latency-associatedpeptide (LAP) and a mature TGFB1 peptide. The mature peptide may alsoform heterodimers with other TGFB family members. This gene isfrequently up-regulated in tumor cells, and mutations in this generesult in Camurati-Engelmann disease. It is not previously known to beinvolved in thyroid function. CSTD encodes a lysosomal aspartylprotease. This proteinase, which is a member of the peptidase C1 family,has a specificity similar to but narrower than that of pepsin A.Transcription of this gene is initiated from several sites, includingone which is a start site for an estrogen-regulated transcript.Mutations in this gene are involved in the pathogenesis of severaldiseases, including breast cancer and possibly Alzheimer disease. Thisgene is not previously known to be involved in thyroid function.

Genes which are down-regulated in both blood and thyroid tissue ofhyperthyroid felines include:

TABLE B C13orf27 chromosome 13 open reading frame 27 GP1BB glycoproteinIb (platelet), beta polypeptide IGLL1 immunoglobulin lambda-likepolypeptide 1 LRRC4B leucine rich repeat containing 4B MALT1 mucosaassociated lymphoid tissue lymphoma translofelineion gene 1 REV3LREV3-like, felinealytic subunit of DNA polymerase zeta (yeast) GNL3guanine nucleotide binding protein-like 3 (nucleolar) RNPC3 RNA-bindingregion (RNP1, RRM) containing 3 LMO4 LIM domain only 4 RFXAP regulatoryfactor X-associated protein MPP6 membrane protein, palmitoylated 6(MAGUK p55 subfamily member 6) DTWD1 DTW domain containing 1 MYC v-mycmyelocytomatosis viral oncogene homolog (avian) TCEA3 transcriptionelongation factor A (SII), 3 RPAP2 RNA polymerase II associated protein2 ZNF292 zinc finger protein 292 CSGALNACT1 chondroitin sulfateN-acetylgalactosaminyltransferase 1 ZFP37 zinc finger protein 37 homolog(mouse) IGJ immunoglobulin J polypeptide, linker protein forimmunoglobulin alpha and mu polypeptides LTBP1 latent transforminggrowth factor beta binding protein 1 ABCA5 ATP-binding cassette,sub-family A (ABC1), member 5 GPRASP2 G protein-coupled receptorassociated sorting protein 2 ARMCX1 armadillo repeat containing,X-linked 1 SEPP1 selenoprotein P, plasma, 1 DCN decorin

Thus for example, downregulation of the genes on this table B can serveas biomarkers for hyperthyroidism. For example, the protein encoded bythe DCN gene is a small cellular or pericellular matrix proteoglycanthat is closely related in structure to biglycan protein. It is acomponent of connective tissue, binds to type I collagen fibrils andplays a role in matrix assembly. It is capable of suppressing the growthof various tumor cell lines. This gene is a candidate gene for Marfansyndrome. It is not previously known to be related to thyroid function.SEPP1 is a secreted protein and is unusual in that it contains 10selenocysteine residues per polypeptide, constituting most of theselenium in plasma. It has been implicated as an extracellularantioxidant and in the transport of selenium, but is not previouslyknown to be involved in thyroid function.

The invention provides, therefore, a method (Method 1) of diagnosinghyperthyroidism or measuring risk of, or predisposition to,hyperthyroidism in a feline, by measuring the expression level of one ormore biomarkers in said feline, said one or more biomarkers selectedfrom the group consisting of the genes listed on Table A (e.g., TGFB1and/or CSTD) and B (e.g., DCN and/or SEPP1), or any of the followinggenes: IYD, TG, SLC5A5, NIS, TPO, TSHR, DUOX1 and DUOX2 (ThOX); theexpression products of these genes; and any combinations thereof,wherein altered expression of one or more, e.g., at least three, forexample five or more, e.g. at least ten, of said biomarkers relative toa normal population or altered expression relative to the feline'sindividual baseline indicates hyperthyroidism or an increased risk of orpredisposition to hyperthyroidism, e.g, according to any of thefollowing methods

-   -   1.1. Method 1 wherein the level of expression of the one or more        biomarkers is determined using either (i) a DNA microarray        comprising one or more oligonucleotides complementary to mRNA or        cDNA corresponding to the one or more marker genes to be        measured, or (ii) a quantitative polymerase chain reaction with        oligonucleotide primers for mRNA or cDNA corresponding to the        one or more marker genes to be measured, e.g.        -   a. The foregoing method wherein the step of measuring gene            expression of one or more biomarkers comprises (i) isolating            RNA from the tissue sample, (ii) reverse transcribing the            RNA to obtain the corresponding cDNA, (iii) isolating and            fragmenting the cDNA thus obtained, (iv) contacting the cDNA            fragments with a DNA microarray comprising one or more            oligonucleotides complementary to cDNA corresponding to the            one or more biomarkers to be measured, and (v) detecting            hybridization between the cDNA fragments and the one or more            oligonucleotides in the DNA microarray.        -   b. Any of the preceding methods involving detecting            hybridization wherein the hybridization between the cDNA            fragments and the one or more oligonucleotides in the DNA            microarray is under stringent conditions.    -   1.2. Method 1 wherein the level of expression of the biomarker        is detected by an antibody to the expressed protein.        -   a. Method 1.2 wherein the biomarker is detected by an            immunoassay selected from a competitive binding assay, a            non-competitive binding assay, a radioimmunoassay, an enzyme            linked immunosorbent assay (ELISA), a sandwich assay, a            precipitin reaction, a gel diffusion immunodiffusion assay,            an agglutination assay, a fluorescent immunoassay,            chemiluminescence immunoassay, immunoPCR immunoassay, a            protein A or protein G immunoassay and an            immunoelectrophoresis assay.        -   b. The foregoing method which is an enzyme-linked            immunosorbent assay (ELISA).        -   c. Method 1.2 wherein the assay is a lateral flow            immunochromatographic assay.        -   d. Method 1.2 wherein the biological sample is blood or            urine.    -   1.3. Method 1 wherein the level of expression of the biomarker        is detected by quantitative mass spectroscopy measuring the        expressed protein in the biological sample, e.g., wherein the        biological sample is blood or urine.    -   1.4. Method 1 wherein the level of expression of the biomarker        is detected by an aptamer recognizing the expressed protein.        -   a. Method 1.4 wherein the aptamer is an oligonucleotide.        -   b. Method 1.4 wherein the aptamer is a peptide.        -   c. Method 1.4 wherein the biological sample is blood or            urine.    -   1.5. Any of the preceding methods wherein the level of        expression of the one or more biomarkers in the biological        sample relative to a control value for expression in normal        sample is greater than 1.25 fold, e.g., at least 30% change.    -   1.6. Any of the preceding methods wherein the level of        expression of the one or more biomarkers in the biological        sample is normalized relative to expression of one or more genes        known to have relatively constant expression.    -   1.7. Any of the preceding methods wherein the biological sample        is a sample of thyroid tissue.    -   1.8. Any of the preceding methods wherein the biological sample        is blood.    -   1.9. Any of the preceding methods further comprising providing        to a feline diagnosed with hyperthyroidism, or an increased risk        of or predisposition to hyperthyroidism, a diet which is        restricted in one or more of iodine, selenium or arachidonic        acid, e.g., a diet wherein the level of iodine is less than 0.35        mg/kg, e.g., 0.01-0.25 mg/kg, the level of selenium is less than        0.1 mg/kg, e.g., 0.01-0.05 mg/kg, and/or the level of        arachidonic acid is less than or equal to 0.02%, e.g.,        0.005-0.01%.    -   1.10. Any of the preceding methods further comprising        administering to a feline diagnosed with hyperthyroidism, or an        increased risk of or predisposition to hyperthyroidism, a        treatment to reduce thyroid hormone production, e.g.,        administering an effective amount of a drug which inhibits        production of T-4, e.g., methimazole or carbimazole, and/or        administering radioiodine therapy, and/or performing a surgical        thyroidectomy.

In a further embodiment, the invention provides reagents, optionallylabeled, useful in the detection of the level of expression of one ormore biomarkers selected from the group consisting of IYD, TG, SLC5A5,NIS, TPO, TSHR, DUOX1, or DUOX2 (ThOX), or from Table A or B, e.g.,TGFB1, CSTD, DCN or SEPP1, and the expression products thereof, e.g.,

-   -   a. Antibodies, for example monoclonal antibodies, single chain        antibodies, and functional antibody fragments, recognizing        feline proteins selected from the group consisting of the        expression products of IYD, TG, SLC5A5, NIS, TPO, TSHR, DUOX1,        or DUOX2 (ThOX), or genes from Table A or B, e.g., TGFB1, CSTD,        DCN or SEPP1.    -   b. Aptamers, for example nucleic acid or peptidic aptamers,        recognizing feline proteins selected from the group consisting        of the expression products of IYD, TG, SLC5A5, NIS, TPO, TSHR,        DUOX1, or DUOX2 (ThOX), or genes from Table A or B, e.g., TGFB1,        CSTD, DCN or SEPP1.    -   c. Isolated and purified or recombinant feline protein selected        from the group consisting of the expression products of IYD, TG,        SLC5A5, NIS, TPO, TSHR, DUOX1, or DUOX2 (ThOX), or genes from        Table A or B, e.g., TGFB1, CSTD, DCN or SEPP1.    -   d. Oligonucleotide probes capable of hybridizing to a feline        gene selected from the group consisting of IYD, TG, SLC5A5, NIS,        TPO, TSHR, DUOX1, or DUOX2 (ThOX), or genes from Table A or B,        e.g., TGFB1, CSTD, DCN or SEPP1.    -   e. In a further embodiment, the invention provides a kit (Kit 1)        for the diagnosis, prognosis or monitoring a thyroid disorder in        a feline, comprising        -   i. means for measuring gene expression of one or more            biomarkers selected from the group consisting of IYD, TG,            SLC5A5, NIS, TPO, TSHR, DUOX1, or DUOX2 (ThOX), or from            Table A or B, e.g., TGFB1, CSTD, DCN or SEPP1, and the            expression products thereof, in a biological sample from the            feline, and        -   ii. instructions for using such means to measure expression            of the one or more biomarkers in a biological sample from            the feline and evaluating the risk, predisposition or            presence of a process leading to a thyroid disorder in the            feline, e.g.    -   1.1 Kit 1 wherein the means for measuring the one or more        biomarkers is one or more nucleic acid probes capable of        detecting gene expression of the one or more biomarkers;    -   1.2 Any of the preceding kits comprising a DNA microarray        comprising one or more nucleic acid probes capable of detecting        gene expression of the one or more biomarkers.    -   1.3 Kit 1 wherein the means for measuring the one or more        biomarkers is one or more antibodies capable of detecting gene        expression of the one or more biomarkers by recognizing the        expressed protein.    -   1.4 Kit 1.3 in ELISA format comprising antibody capable of        detecting the one or more biomarkers; isolated, purified or        recombinant protein corresponding to the expressed protein; and        buffer.    -   1.5 Kit 1 wherein the means for measuring the one or more        biomarkers is one or more aptamers, e.g., as hereinbefore        described, capable of detecting gene expression of the one or        more biomarkers by recognizing the expressed protein.    -   1.6 Any of the foregoing kits adapted for use in any of the        foregoing Method 1 et seq.

The invention further provides the use of

-   -   a nucleotide sequence corresponding to or complementary to a        gene for IYD, TG, SLC5A5, NIS, TPO, TSHR, DUOX1, or DUOX2        (ThOX), or from Table A or B, e.g., TGFB1, CSTD, DCN or SEPP1,    -   or of an antibody to a protein selected from IYD, TG, SLC5A5,        NIS, TPO, TSHR, DUOX1, or DUOX2 (ThOX), or from Table A or B,        e.g., TGFB1, CSTD, DCN or SEPP1, or    -   of an aptamer to a protein selected from IYD, TG, SLC5A5, NIS,        TPO, TSHR, DUOX1, or DUOX2 (ThOX), or from Table A or B, e.g.,        TGFB1, CSTD, DCN or SEPP1, or    -   isolated, purified or recombinant feline protein selected from        IYD, TG, SLC5A5, NIS, TPO, TSHR, DUOX1, or DUOX2 (ThOX), or from        Table A or B, e.g., TGFB1, CSTD, DCN or SEPP1,    -   in a method according to Method 1, et seq., or    -   in the manufacture of a kit according to Kit 1, et seq.

Further areas of applicability of the present invention will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whileindicating the preferred embodiment of the invention, are intended forpurposes of illustration only and are not intended to limit the scope ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

The following description of the preferred embodiments is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses.

Certain Definitions

The term “antibody” means any immunoglobulin that binds to a specificantigen, including IgG, IgM, IgA, IgD, and IgE antibodies. The termincludes polyclonal, monoclonal, monovalent, humanized, heteroconjugate,antibody compositions with polyepitopic specificity, chimeric,bispecific antibodies, diabodies, single-chain antibodies, and antibodyfragments such as Fab, Fab′, F(ab′)₂, and Fv, or other antigen-bindingfragments.

The term “array” means an ordered arrangement of at least two probes ona substrate. At least one of the probes is a control or standard and atleast one of the probes is a diagnostic probe. The arrangement of fromabout two to about 40,000 probes on a substrate assures that the sizeand signal intensity of each labeled complex formed between a probe anda sample polynucleotide or polypeptide is individually distinguishable.The collection of molecules deposited on the array may be preparedeither synthetically or biosynthetically. The array may take a varietyof forms including libraries of soluble molecules, libraries ofcompounds tethered to resin beads, silica chips or other solid supports.The nucleic acid array may include libraries of nucleic acids which canbe prepared by spotting nucleic acids in essentially any length (forexample, from 1 to about 1,000 nucleotides in length) onto a substrate.A nucleic acid probe array preferably comprises nucleic acids bound to asubstrate in known locations. In other embodiments, the system mayinclude a solid support or substrate, such as a membrane, filter,microscope slide, microwell, sample tube, bead, bead array, or the like.The solid support may be made of various materials, including paper,cellulose, nylon, polystyrene, polycarbonate, plastics, glass, ceramic,stainless steel, or the like. The solid support may preferably have arigid or semi-rigid surface, and may preferably be spherical (e.g.,bead) or substantially planar (e.g., flat surface) with appropriatewells, raised regions, etched trenches, or the like. The solid supportmay also include a gel or matrix in which nucleic acids may be embedded.

The term “biomarkers” refers to genes and gene products encoded by agene of the invention or a homolog thereof, especially a feline homologthereof, wherein the gene has been determined to have beendifferentially expressed as a result of a disease, condition, disorderor the administration of a substance, drug, nutrient or dietarycomponent or combinations thereof. A biomarker may be a polynucleotide,polypeptide, protein, RNA, including an RNA transcript or itstranslation product, DNA, cDNA, a metabolite of one or more of theforegoing molecules, or a useful variant of any one of the foregoingmolecules, the differential expression of which is associated with athyroid disorder, and wherein the correlation of such differentialexpression in a sample taken from a test animal to a sample taken from acontrol animal can be used in the diagnosis, prognosis, monitoring ortreatment of condition, disease or disorder in an animal in needthereof. In addition, a biomarker can be generally used to refer to anyportion or segment of such gene or protein that can identify orcorrelate with the full-length gene or protein, for example, in an assayor other method of the invention. Biomarker expression can also beidentified by detection of biomarker translation (i.e., detection ofbiomarker protein in a sample). Methods suitable for the detection ofbiomarker protein include any suitable method for detecting and/ormeasuring proteins from a cell or cell extract. Such methods include,but are not limited to, immunoblot (e.g., Western blot), enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (MA), immunoprecipitation,immunohistochemistry and immunofluorescence. Particularly preferredmethods for detection of proteins include any single-cell assay,including immunohistochemistry and immunofluorescence assays. Suchmethods are well known in the art. Furthermore, antibodies againstcertain of the biomarkers described herein are known in the art and aredescribed in the public literature, and methods for their preparationare well known to the skilled worker.

The term “comparably” as used to compare expression of a test sample toa control sample shall mean indicia of like character and quantity andshall include, without limitation, values within one standard deviationaround the mean value to which said comparison is made and valuesencompassing differential expression between the test sample and controlsample.

The terms “differentially expressed gene,” “differential geneexpression,” “differential expression” or “differentially expressed” andtheir synonyms, which are used interchangeably, refer to a gene whoseexpression is activated to a higher or lower level in a subjectsuffering from a disease, condition, or disorder, or as a result of thebeing administered a substance, drug, nutrient or dietary component orcombinations thereof, relative to its expression in a normal or controlsubject. The terms also include genes whose expression is activated to ahigher or lower level at different stages of the same disease. It isalso understood that a differentially expressed gene may be eitheractivated or inhibited at the nucleic acid level or protein level, ormay be subject to alternative splicing to result in a differentpolypeptide product. Such differences may be evidenced by a change inmRNA levels, surface expression, secretion or other partitioning of apolypeptide, for example. Differential gene expression may include acomparison of expression between two or more genes or their geneproducts, or a comparison of the ratios of the expression between two ormore genes or their gene products, or even a comparison of twodifferently processed products of the same gene, which differ betweennormal subjects and subjects suffering from a disease, condition, ordisorder, or as a result of the being administered a substance, drug,nutrient or dietary component or combinations thereof, or betweenvarious stages of the same disease, condition, or disorder, or as aresult of being administered different amounts of a substance, drug,nutrient or dietary component or combinations thereof. Differentialexpression includes both quantitative, as well as qualitative,differences in the temporal or cellular expression pattern in a gene orits expression products among, for example, normal and diseased cells,or among cells which have undergone different disease events or diseasestages. For the purpose of this invention, “differential geneexpression” is considered to be present when there is at least an about2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or 1.0-fold, preferablyat least about two-fold or more, more preferably at least about 2.5, 3or 4 or more fold change in the amount of transcribed polynucleotides ortranslated protein in a sample.

The term “fold” when used as a measure of differential gene expressionmeans an amount of gene expression in a feline that is a multiple or afraction of gene expression compared to the amount of gene expression ina comparison feline, e.g., a feline having symptoms related tohyperthyroidism, at risk for or having hyperthyroidism compared to ananimal not demonstrating such a condition. For example, a gene that isexpressed 2 times as much in the animal as in the comparison animal hasa 2-fold differential gene expression and a gene that is expressedone-half as much in the animal as in the comparison animal also has a2-fold differential gene expression.

The term “fragment” means (1) an oligonucleotide or polynucleotidesequence that is a portion of a complete sequence and that has the sameor similar activity for a particular use as the complete polynucleotidesequence or (2) a peptide or polypeptide sequence that is a portion of acomplete sequence and that has the same or similar activity for aparticular use as the complete polypeptide sequence. Such fragments cancomprise any number of nucleotides or amino acids deemed suitable for aparticular use. Generally, oligonucleotide or polynucleotide fragmentscontain at least about 10, 50, 100, or 1000 nucleotides and polypeptidefragments contain at least about 4, 10, 20, or 50 consecutive aminoacids from the complete sequence. The term encompasses polynucleotidesand polypeptides variants of the fragments. A polynucleotide, forexample, can be broken up, or fragmented into, a plurality of segments.

Various methods of fragmenting nucleic acid are well known in the art.These methods may be, for example, either chemical or physical innature. Chemical fragmentation may include partial degradation with aDNase; partial depurination with acid; the use of restriction enzymes;intron-encoded endonucleases; DNA-based cleavage methods, such astriplex and hybrid formation methods, that rely on the specifichybridization of a nucleic acid segment to localize a cleavage agent toa specific location in the nucleic acid molecule; or other enzymes orcompounds which cleave DNA at known or unknown locations. Physicalfragmentation methods may involve subjecting the DNA to a high shearrate. High shear rates may be produced, for example, by moving DNAthrough a chamber or channel with pits or spikes, or forcing the DNAsample through a restricted size flow passage, e.g., an aperture havinga cross sectional dimension in the micron or submicron scale. Otherphysical methods include sonication and nebulization. Combinations ofphysical and chemical fragmentation methods may likewise be employedsuch as fragmentation by heat and ion-mediated hydrolysis. See forexample, Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 3rdEd. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)(“Sambrook et al.), which is incorporated herein by reference for allpurposes. These methods can be optimized to digest a nucleic acid intofragments of a selected size range. Useful size ranges may be from 100,200, 400, 700 or 1000 to 500, 800, 1500, 2000, 4000 or 10,000 basepairs. However, larger size ranges such as 4000, 10,000 or 20,000 to10,000, 20,000 or 500,000 base pairs may also be useful.

The term “gene” or “genes” means a complete or partial segment of DNAinvolved in producing a polypeptide, including regions preceding andfollowing the coding region (leader and trailer) and interveningsequences (introns) between individual coding segments (exons). The termencompasses any DNA sequence that hybridizes to the complement of genecoding sequences.

The term “homolog” means (1) a polynucleotide, including polynucleotidesfrom the same or different animal species, having greater than 30%, 50%,70%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequencesimilarity to a polynucleotide and having the same or substantially thesame properties and performing the same or substantially the samefunction as the complete polynucleotide, or having the capability ofspecifically hybridizing to a polynucleotide under stringent conditionsor (2) a polypeptide, including polypeptides from the same or differentanimal species, having greater than 30%, 50%, 70%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence similarity to a polypeptideidentified by the expression of polynucleotides and having the same orsubstantially the same properties and performing the same orsubstantially the same function as the complete polypeptide, or havingthe capability of specifically binding to a polypeptide identified bythe expression of polynucleotides. Sequence similarity of twopolypeptide sequences or of two polynucleotide sequences is determinedusing methods known to skilled artisans, e.g., the algorithm of Karlinand Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410 (1990)). To obtain gappedalignments for comparison purposes, Gapped Blast can be utilized asdescribed in Altschul et al. (Nucl. Acids Res. 25: 3389-3402 (1997)).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) are used. Seehttp://www.ncbi.nlm.nih.gov.

The term “hybridization” refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide. The term “hybridization” may also referto triple-stranded hybridization. The resulting (usually)double-stranded polynucleotide is a “hybrid.” The proportion of thepopulation of polynucleotides that forms stable hybrids is referred toherein as the “degree of hybridization.”

Hybridization reactions can be performed in absolute or differentialhybridization formats. In the absolute hybridization format,polynucleotides derived from one sample are hybridized to the probes ina nucleic acid array. Signals detected after the formation ofhybridization complexes correlate to the polynucleotide levels in thesample. In the differential hybridization format, polynucleotidesderived from two samples are labeled with different labeling moieties. Amixture of these differently labeled polynucleotides is added to anucleic acid array. The nucleic acid array is then examined underconditions in which the emissions from the two different labels areindividually detectable. In one embodiment, the fluorophores Cy3 and Cy5(Amersham Pharmacia Biotech, Piscataway, N.J.) are used as the labelingmoieties for the differential hybridization format.

Signals gathered from nucleic acid arrays can be analyzed usingcommercially available software, such as those provided by Affymetrix orAgilent Technologies. Controls, such as for scan sensitivity, probelabeling and cDNA or cRNA quantization, are preferably included in thehybridization experiments. Hybridization signals can be scaled ornormalized before being subject to further analysis. For instance,hybridization signals for each individual probe can be normalized totake into account variations in hybridization intensities when more thanone array is used under similar test conditions. Hybridization signalscan also be normalized using the intensities derived from internalnormalization controls contained on each array. In addition, genes withrelatively consistent expression levels across the samples can be usedto normalize the expression levels of other genes. In one embodiment,probes for certain maintenance genes are included in a nucleic acidarray of the present invention. These genes are chosen because they showstable levels of expression across a diverse set of tissues.Hybridization signals can be normalized and/or scaled based on theexpression levels of these maintenance genes.

The term “hybridization complex” means a complex that is formed betweensample polynucleotides when the purines of one polynucleotide hydrogenbond with the pyrimidines of the complementary polynucleotide, e.g.,5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′. The degree ofcomplementarily and the use of nucleotide analogs affect the efficiencyand stringency of hybridization reactions.

The term “hybridization probes” includes nucleic acids (such asoligonucleotides) capable of binding in a base-specific manner to acomplementary strand of nucleic acid. Such probes include peptidenucleic acids, as described in Nielsen et al., Science 254:1497-1500(1991), Nielsen Curr. Opin. Biotechnol., 10:71-75 (1999) and othernucleic acid analogs and nucleic acid mimetics. See U.S. Pat. No.6,156,501 filed Apr. 3, 1996.

“Nucleic acid sequence” means an oligonucleotide, nucleotide orpolynucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be single- or double-stranded, andrepresent the sense or antisense strand.

The term “polynucleotide” or “oligonucleotide” means a polymer ofnucleotides. The term encompasses DNA and RNA (including cDNA and mRNA)molecules, either single or double stranded and, if single stranded, itscomplementary sequence in either linear or circular form. The term alsoencompasses fragments, variants, homologs, and alleles, as appropriatefor the sequences that have the same or substantially the sameproperties and perform the same or substantially the same function asthe original sequence. The sequences may be fully complementary (nomismatches) when aligned or may have up to about a 30% sequencemismatch. Preferably, for polynucleotides, the chain contains from about20 to 10,000 nucleotides, more preferably from about 150 to 3,500nucleotides. Preferably, for oligonucleotides, the chain contains fromabout 2 to 100 nucleotides, more preferably from about 6 to 30nucleotides. The exact size of a polynucleotide or oligonucleotide willdepend on various factors and on the particular application and use ofthe polynucleotide or oligonucleotide. The term includes nucleotidepolymers that are synthesized and that are isolated and purified fromnatural sources. The term “polynucleotide” is inclusive of“oligonucleotide.”

The term “polypeptide,” “peptide,” or “protein” means a polymer of aminoacids. The term encompasses naturally occurring and non-naturallyoccurring (synthetic) polymers and polymers in which artificial chemicalmimetics are substituted for one or more amino acids. The term alsoencompasses fragments, variants, and homologs that have the same orsubstantially the same properties and perform the same or substantiallythe same function as the original sequence. The term encompass polymersof any length, preferably polymers containing from about 2 to 1000 aminoacids, more preferably from about 5 to 500 amino acids. The termincludes amino acid polymers that are synthesized and that are isolatedand purified from natural sources.

The term “probe” means (1) an oligonucleotide or polynucleotide, eitherRNA or DNA, whether occurring naturally as in a purified restrictionenzyme digest or produced synthetically, that is capable of annealingwith or specifically hybridizing to a polynucleotide with sequencescomplementary to the probe or (2) a peptide or polypeptide capable ofspecifically binding a particular protein or protein fragment to thesubstantial exclusion of other proteins or protein fragments. Anoligonucleotide or polynucleotide probe may be either single or doublestranded. The exact length of the probe will depend upon many factors,including temperature, source, and use. For example, for diagnosticapplications, depending on the complexity of the target sequence, anoligonucleotide probe typically contains about 10 to 100, 15 to 50, or15 to 25 nucleotides. In certain diagnostic applications, apolynucleotide probe contains about 100-1000, 300-600, nucleotides,preferably about 300 nucleotides. The probes herein are selected to be“substantially” complementary to different strands of a particulartarget sequence. This means that the probes must be sufficientlycomplementary to specifically hybridize or anneal with their respectivetarget sequences under a set of predetermined conditions. Therefore, theprobe sequence need not reflect the exact complementary sequence of thetarget. For example, a noncomplementary nucleotide fragment may beattached to the 5′ or 3′ end of the probe, with the remainder of theprobe sequence being complementary to the target sequence.Alternatively, noncomplementary bases or longer sequences can beinterspersed into the probe provided that the probe sequence hassufficient complementarity with the sequence of the targetpolynucleotide to specifically anneal to the target polynucleotide. Apeptide or polypeptide probe may be any molecule to which the protein orpeptide specifically binds, including DNA (for DNA binding proteins),antibodies, cell membrane receptors, peptides, cofactors, lectins,sugars, polysaccharides, cells, cell membranes, organelles andorganellar membranes.

The terms “sample” and “specimen” mean any animal tissue or fluidcontaining polynucleotides, including cells and other tissue containingDNA and RNA. Examples include: blood, kidney, connective, epithelial,lymphoid, muscle, nervous, sputum, and the like. A sample may be solidor liquid and that may contain DNA, RNA, cDNA, for example, bodilyfluids such as blood or urine, cells, cell preparations or solublefractions or media aliquots thereof, chromosomes, organelles, and thelike.

The term “specifically bind” means a special and precise interactionbetween two molecules which is dependent upon their structure,particularly their molecular side groups. For example, the intercalationof a regulatory protein into the major groove of a DNA molecule, thehydrogen bonding along the backbone between two single stranded nucleicacids, or the binding between an epitope of a protein and an agonist,antagonist, or antibody.

The term “specifically hybridize” means an association between twosingle stranded polynucleotides of sufficiently complementary sequenceto permit such hybridization under predetermined conditions generallyused in the art (sometimes termed “substantially complementary”). Forexample, the term may refer to hybridization of a polynucleotide probewith a substantially complementary sequence contained within a singlestranded DNA or RNA molecule according to an aspect of the invention, tothe substantial exclusion of hybridization of the polynucleotide probewith single stranded polynucleotides of non-complementary sequence.

The term “stringent conditions” means (1) hybridization in 50% (vol/vol)formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750mM NaCl, 75 mM sodium citrate at 42° C., (2) hybridization in 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 mg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C.; with washes at 42° C. in 0.2×SSC and 0.1% SDS or washes with0.015 M NaCl, 0.0015 M sodium citrate, 0.1% Na2SO4 at 50° C. or similarart-recognized procedures employing similar low ionic strength and hightemperature washing agents and similar denaturing agents.

The term “useful variations” means (1) for a polynucleotide, thecomplements of the polynucleotide; the homologs of the polynucleotideand its complements; the variants of the polynucleotide, itscomplements, and its homologs; and the fragments of the polynucleotide,its complements, its homologs, and its variants and (2) for apolypeptide, the homologs of the polypeptide; the variants of thepolypeptide and its homologs; and the fragments of the polynucleotide,its homologs, and its variants.

The term “variant” means (1) a polynucleotide sequence containing anysubstitution, variation, modification, replacement, deletion, oraddition of one or more nucleotides from or to a polynucleotide sequenceand that has the same or substantially the same properties and performsthe same or substantially the same function as the original sequence and(2) a polypeptide sequence containing any substitution, variation,modification, replacement, deletion, or addition of one or more aminoacids from or to a polypeptide sequence and that has the same orsubstantially the same properties and performs the same or substantiallythe same function as the original sequence. The term therefore includessingle nucleotide polymorphisms (SNPs) and allelic variants and includesconservative and non-conservative amino acid substitutions inpolypeptides. The term also encompasses chemical derivatization of apolynucleotide or polypeptide and substitution of nucleotides or aminoacids with nucleotides or amino acids that do not occur naturally, asappropriate.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention.

Probes

The probes useful in the practice of the invention and which areutilized in the identification of the feline biomarkers in the felinesamples correspond to the following probe identification numbers used inthe proprietary feline gene chip manufactured by Affymetrix, identifiedas Affymetrix Feline GeneChip®, as more fully described in thisspecification.

Other and further objects, features, and advantages of the presentinvention will be readily apparent to those skilled in the art.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 5 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 2 to 4, from2 to 5, from 3 to 5 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, and 5. This applies regardless of thebreadth of the range

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as: Genome Analysis: A Laboratory Manual Series(Vols. I-IV); Using Antibodies: A Laboratory Manual; Cells: A LaboratoryManual; PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press);Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York; Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000); Lehninger, Principles of Biochemistry 3rdEd., W.H. Freeman Pub., New York, N.Y; and Berg et al. (2002)Biochemistry, 5th Ed., W.H. Freeman Pub., New York, N.Y., all of whichare herein incorporated in their entirety by reference for all purposes.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248,6,309,822 and 6,344,316. Genotyping and uses therefore are shown in U.S.Ser. No. 60/319,253, 10/013,598, and U.S. Pat. Nos. 5,856,092,6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179.Other uses are embodied in U.S. Pat. Nos. 5,871,928, 5,902,723,6,045,996, 5,541,061, and 6,197,506.

Those skilled in the art will recognize that the products and methodsembodied in the present invention may be applied to a variety ofsystems, including commercially available gene expression monitoringsystems involving nucleic acid probe arrays, membrane blots, microwells,beads and sample tubes, constructed with various materials using variousmethods known in the art. Accordingly, the present invention is notlimited to any particular environment, and the following description ofspecific embodiments of the present invention is for illustrativepurposes only.

The gene expression monitoring system, in a preferred embodiment, maycomprise a nucleic acid probe array (including an oligonucleotide array,a cDNA array, a spotted array, and the like), membrane blot (such asused in hybridization analysis such as Northern, Southern, dot, and thelike), or microwells, sample tubes, beads or fibers (or any solidsupport comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722,5,744,305, 5,677,195, 5,445,934 and 6,040,193 which are incorporatedherein by reference. The gene expression monitoring system may alsocomprise nucleic acid probes in solution.

The present invention also contemplates sample preparation involvingamplification. A genomic sample may be amplified by a variety ofmechanisms, some of which may employ PCR. See, e.g., PCR Technology:Principles and Applications for DNA Amplification (Ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods andApplications (Eds. Innis, et al., Academic Press, San Diego, Calif.,1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert etal., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson etal., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195,4,800,159 4,965,188, and 5,333,675, and each of which is incorporatedherein by reference in their entireties for all purposes. The sample maybe amplified on the array. See, for example, U.S. Pat. No. 6,300,070 andU.S. patent application Ser. No. 09/513,300, which are incorporatedherein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517 and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617, 6,344,316and in U.S. Ser. No. 09/854,317, each of which is incorporated herein byreference.

Additional methods of sample preparation and techniques are described inDong et al., Genome Research 11, 1418 (2001), in U.S. Pat. Nos.6,361,947, 6,391,592 and U.S. patent application Ser. Nos. 09/916,135,09/920,491, 09/910,292 and 10/013,598.

The gene expression monitoring system according to the present inventionmay be used to facilitate a comparative analysis of expression indifferent cells or tissues, different subpopulations of the same cellsor tissues, different physiological states of the same cells or tissue,different developmental stages of the same cells or tissue, or differentcell populations of the same tissue. In a preferred embodiment, theproportional amplification methods of the present invention can providereproducible results (i.e., within statistically significant margins oferror or degrees of confidence) sufficient to facilitate the measurementof quantitative as well as qualitative differences in the testedsamples.

Polynucleotide hybridization assays are well known in the art.Hybridization assay procedures and conditions will vary depending on theapplication and are selected in accordance with the general bindingmethods known including those referred to in: Maniatis et al. MolecularCloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989);Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to MolecularCloning Techniques (Academic Press, Inc., San Diego, Calif., 1987);Young and Davis, P.N.A.S, 80: 1194 (1983). Methods and apparatus forcarrying out repeated and controlled hybridization reactions have beendescribed in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996, 6,386,749and 6,391,623 each of which are incorporated herein by reference. Signaldetection of hybridization between ligands in certain preferredembodiments. See U.S. Pat. Nos. 5,143,854, 5,578,832, 5,631,734,5,834,758, 5,936,324, 5,981,956, 6,025,601, 6,141,096, 6,185,030,6,201,639, 6,218,803 and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes. Methods and apparatus for signaldetection and processing of intensity data are disclosed in, forexample, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734,5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956,6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

The invention is not limited to the particular methodology, protocols,and reagents described herein because they may vary. Further, theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention.

In one embodiment, the present invention encompasses one or more genesor gene segments (“genes” as defined herein) that are differentiallyexpressed in abnormal animals compared to normal animals. The inventionis based upon the discovery of polynucleotides that are differentiallyexpressed in abnormal animals compared to normal animals. The genes areidentified by comparing the expression of genes in tissue samples takenfrom animals diagnosed as abnormal with genes in tissue samples fromanimals diagnosed as normal using Affymetrix GeneChip® technology.

The polynucleotides and genes are identified by measuring differences ingene expression from tissue samples taken from felines diagnosed asabnormal and having a thyroid disorder with gene expression in tissuesamples from felines diagnosed as normal. Changes in gene expression canbe determined by any method known to skilled artisans. Generally,changes in gene expression are determined by measuring transcription(determining the amount of mRNA produced by a gene) or measuringtranslation (determining the amount of protein produced by a gene). Theamount of RNA or protein produced by a gene can be determined using anymethod known to skilled artisans for quantifying polynucleotides andproteins.

Generally, mRNA expression is determined using polymerase chain reaction(PCR) (including, without limitation, reverse transcription-PCR (RT-PCR)and quantitative real-time PCR (qPCR)), short or long oligonucleotidearrays, cDNA arrays, EST sequencing, Northern blotting, SAGE, MPSS, MS,bead arrays and other hybridization methods. The RNA measured istypically in the form of mRNA or reverse transcribed mRNA.

Protein or polypeptide expression is determined using variouscolorimetric and spectroscopic assays and methods such as quantitativeWestern blots, ELISA, 2D-gels, gas or liquid chromatography, mass-spec,the lowry assay, the biuret assay, fluorescence assays, turbidimetricmethods, the bicinchoninic assay, protein chip technology, infraredabsorbance, ninhydrin, the Bradford assay, and ultraviolet absorbance.

Gene chips allow a large-scale study of biological processes and themeasurement of activity within a cell at a certain point in time.Microarray analysis permits one to account for differences in phenotypeson a large-scale genetic basis. Actual measurement of gene expressionproducts is a more accurate indicator of gene function than determiningsequences per se. Microarray analysis is based upon quantifying theconcentration of a gene's mRNA transcript in a cell at a given time. DNAis immobilized on a medium and labeled target mRNA is hybridized withprobes on the array. Binding of the labeled mRNA to the probes ismeasured by laser analysis. The measurement is a count of photonsemitted. The entire chip is scanned and digitally imaged. The image isprocessed to locate probes and to assign intensity measurements to eachprobe. In this manner up- and down-regulated genes may be determined.The analysis enables the skilled person to find groups of genes withsimilar expression profiles and to determine tissues with similarexpression profiles. In this manner, genes that explain the observeddifferences in tissue samples can be identified.

Affymetrix Gene Chips typically employ probes of 25 bp and probe sets of11 to 20 probes corresponding to a particular gene or EST. The chip isconstructed with a perfect match and mismatch probe of 25 bp each, theformer being perfectly complementary to a specific region of a gene andthe latter having the 13^(th) bp substituted to make a mismatch. A probesummarization algorithm is used to determine background correction,normalization and probe summarization, which is the conversion of probevalues to probe set expression values. RMA is one of the algorithms thatmay be used for this purpose. The algorithm performs the last two stepsof analysis, normalization and summarization of probe-level intensitymeasurements. The perfect match values are, therefore, backgroundcorrected, normalized and summarized into a set of expressionmeasurements.

The raw data is analyzed using GeneSpring version 7.0 (GS) software(Agilent Corporation) and validated using the R-Bioconductor (RB)freeware. Both software packages are used to compute probe intensitiesfrom the CEL files generated by the Affymetrix Instrument. ThePresent/Absent/Marginal calls per probe and P-values are computed usingthe R-Bioconductor and GeneSpring software separately.

Generally, differential gene expression in abnormal animals compared tonormal animals is determined by measuring the expression of at least onegene. Preferably, the expression of two or more differentially expressedgenes is measured to provide a gene expression pattern or geneexpression profile. More preferably, the expression of a plurality ofdifferentially expressed genes is measured to provide additionalinformation for a more significant gene expression pattern or profile.

The present invention provides a plurality of markers that together oralone are or can be used as markers of renal disease. In especiallyuseful embodiments of the invention, a plurality of these markers can beselected and their mRNA expression may be measured simultaneously toprovide expression profiles for use in various aspects of the inventionsdescribed in this application.

In another aspect, the invention provides a device suitable fordetecting the expression of a plurality of genes differentiallyexpressed in abnormal felines compared to normal felines. The devicecomprises a substrate having a plurality of the oligonucleotide orpolynucleotide probes of the present invention affixed to the substrateat known locations. The device is essentially an immobilized version ofthe oligonucleotide or polynucleotide probes described herein. Thedevice is useful for rapid and specific detection of genes andpolynucleotides and their expression patterns and profiles. Typically,such probes are linked to a substrate or similar solid support and asample containing one or more polynucleotides (e.g., a gene, a PCRproduct, a ligase chain reaction (LCR) product, a DNA sequence that hasbeen synthesized using amplification techniques, or a mixture thereof)is exposed to the probes such that the sample polynucleotide(s) canhybridize to the probes. The probes, the sample polynucleotide(s), orboth, are labeled, typically with a fluorophore or other tag such asstreptavidin, and detected using methods known to skilled artisans. Ifthe sample polynucleotide(s) is labeled, hybridization may be detectedby detecting bound fluorescence. If the probes are labeled,hybridization is typically detected by label quenching. If both theprobe and the sample polynucleotide(s) are labeled, hybridization istypically detected by monitoring a color shift resulting from proximityof the two bound labels. A variety of labeling strategies and labels areknown to skilled artisans, particularly for fluorescent labels.Preferably, the probes are immobilized on substrates suitable forforming an array (known by several names including DNA microarray, genechip, biochip, DNA chip, and gene array) comparable to those known inthe art.

Methods for determining the amount or concentration of protein in asample are known to skilled artisans. Such methods includeradioimmunoassays, competitive-binding assays, Western blot analysis,and ELISA assays. For methods that use antibodies, polyclonal andmonoclonal antibodies are suitable. Such antibodies may beimmunologically specific for a protein, protein epitope, or proteinfragment.

Some embodiments of the invention utilize antibodies for the detectionand quantification of proteins produced by expression of thepolynucleotides of the present invention. Although proteins may bedetected by immunoprecipitation, affinity separation, Western blotanalysis, protein arrays, and the like, a preferred method utilizesELISA technology wherein the antibody is immobilized on a solid supportand a target protein or peptide is exposed to the immobilized antibody.Either the probe, or the target, or both, can be labeled using knownmethods.

In a further aspect, the invention provides a method for detecting thedifferential expression of one or more genes differentially expressed inabnormal felines compared to normal felines in a sample. The methodcomprises (a) hybridizing a combination comprising a plurality ofpolynucleotide probes that are differentially expressed in abnormalfelines compared to normal felines with polynucleotides in the sample toform one or more hybridization complexes; (b) optionally, hybridizing acombination comprising a plurality of polynucleotide probes that aredifferentially expressed in abnormal felines compared to normal felineswith polynucleotides in a standard to form one or more hybridizationcomplexes; (c) detecting the hybridization complexes from the sampleand, optionally, the standard from step (b); and (d) comparing thehybridization complexes from the sample with the hybridization complexesfrom a standard, wherein a difference in the amount of hybridizationcomplexes between the standard and sample indicate differentialexpression of genes differentially expressed in abnormal animalscompared to normal animals in the sample.

Step (b) and part of step (c) are optional and are used if a relativelycontemporaneous comparison of two or more test systems is to beconducted. However, in a preferred embodiment, the standard used forcomparison is based upon data previously obtained using the method.

These probes are exposed to a sample to form hybridization complexesthat are detected and compared with those of a standard. The differencesbetween the hybridization complexes from the sample and standardindicate differential expression of polynucleotides and therefore genesdifferentially expressed in abnormal felines compared to normal felinesin the sample. In a preferred embodiment, probes are made tospecifically detect polynucleotides or fragments thereof produced by oneor more of the genes or gene fragments identified by the presentinvention. Methods for detecting hybridization complexes are known toskilled artisans.

In another aspect, the invention provides a method for detecting thedifferential expression of genes differentially expressed in abnormalfelines compared to normal felines in a sample. The method comprises (a)reacting a combination comprising a plurality of polypeptide probes withproteins in the sample under conditions that allow specific bindingbetween the probes and the proteins to occur, wherein the proteins boundby the probes are differentially expressed in an abnormal felinecompared to a normal feline; (b) optionally, reacting a combinationcomprising a plurality of polypeptide probes with proteins in a standardunder conditions that allow specific binding between the probes and theproteins to occur, wherein the proteins bound by the probes aredifferentially expressed in an abnormal feline compared to a normalfeline; (c) detecting specific binding in the sample and, optionally,the standard from step (b); and (d) comparing the specific binding inthe sample with that of a standard, wherein differences between thespecific binding in the standard and the sample indicate differentialexpression of genes differentially expressed in abnormal felinescompared to normal felines in the sample.

These probes are exposed to a sample to form specific binding that isdetected and compared with those of a standard. The differences betweenthe specific binding from the sample and standard indicate differentialexpression of proteins and therefore genes differentially expressed inabnormal felines compared to normal felines, particularlyabnormal-associated genes, in the sample. In a preferred embodiment,probes are made to specifically detect proteins or fragments thereofproduced by one or more of the genes or gene fragments identified by thepresent invention.

In one embodiment, the method further comprises exposing the feline orsample to a test substance before reacting the polypeptides with theproteins. Then, the comparison is indicative of whether the testsubstance altered the expression of genes differentially expressed inabnormal felines compared to normal felines, particularlyabnormal-associated genes, in the sample.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range. In addition, all references citedherein are hereby incorporated by reference in their entireties. In theevent of a conflict in a definition in the present disclosure and thatof a cited reference, the present disclosure controls.

Example 1—Gene Expression in Hyperthyroid Felines

This study focuses on gene expression in thyroid tissue and whole bloodin felines with hyperthyroidism. All hyperthyroid felines are diagnosedby veterinarians on the basis of clinical signs and elevated T4concentrations. In the tissue study, 1308 significant differentialexpression genes are found between hyperthyroid felines andnon-hyperthyroid felines with FDR=0.1 and fold change of >=+/−1.25. Inthe whole blood study, 1094 significant genes are found betweenhyperthyroid felines and non-hyperthyroid felines with the same cutoffas shown above. In the tissue study, all the genes known to be involvedin the pathway leading to the production of thyroid hormones are foundto be up-regulated in the hyperthyroid felines. The increased expressionof these genes clearly contributes to a high T4 production in the felinebody. In the whole blood study, 60 genes are found to overlap with thosefound from the solid tissue work that appear to be regulated in the samedirection as seen in the tissue study. By using these 60 genes, thehyperthyroid felines can be distinguished from the normal felines astested in the whole blood, as well as in the tissue.

Felines are divided into two groups. One group includes felines with noevidence of disease, and the other group includes felines withhyperthyroidism. Both tissues and whole blood are collected for geneexpression analysis.

RNA Isolation from Solid Tissue

Tissue samples are homogenized using the Ultra-Turrax T25 PowerHomogenizer. RNA is isolated using the protocol outlined in SOP #LAB-LS-028.3. The RNA quality and quantity is determined by using theAgilent 2100 Bioanalyzer (Agilent Technologies) according to themanufacturer's instructions. RNA integrity is determined by using theRNA integrity number (RIN; Agilent 2100 RIN Beta Version Software).Purified RNA samples are stored at −70° C.

Sample Preparation from Whole Blood

Blood is collected and processed according to the manufacturer'sinstructions in PAXgene RNA blood tubes. The PAXgene RNA blood tubes arestored at −20° C. (short-term storage, <6 months) and −70° C. (>6months) before RNA is isolated.

RNA Isolation from Whole Blood

RNA is isolated with the PAXgene Blood RNA Isolation Kit according tothe manufacturer's instructions (Qiagen, p/n 762164). The RNA qualityand quantity is determined using the Agilent 2100 Bioanalyzer (AgilentTechnologies) according to the manufacturer's instructions. RNAintegrity is determined by using the RNA integrity number (RIN; Agilent2100 RIN Beta Version Software). Purified RNA samples are stored at −70°C.

Whole Blood Probe Preparation

Labeling and amplification reagents are obtained from NuGENTechnologies, Inc (San Carlos, Calif., USA) and biotinylated cDNAtargets are prepared according to manufacturer's instructions.Double-stranded cDNA is synthesized from approximately 75 ng of totalRNA, followed by a linear isothermal amplification (SPIA Amplification™)step to produce single-stranded cDNA. Fragmentation is followed by adirect labeling process that attached biotin to the amplified probe.Probe purifications are performed using DNA Clean and Concentrator—25(Zymo Research, Orange, Calif., USA).

Solid Tissue Probe Preparation

Labeling and amplification reagents are obtained from Affymetrix (3420Central Expressway, Santa Clara, Calif. 95051). The One-cycle cDNASynthesis Kit (p/n 900431) and 3′IVT Labeling Kit (p/n 900449) is usedto synthesize, fragment and label each cDNA target. All cDNA targets areprepared according to manufacturer's instructions. Probe purificationsare performed using the GeneChip Sample Cleanup Module (Affymetrix p/n900371). It is important to note that Affymetrix no longer sells theseexact products, per se, as they have reformulated and renamed theirlabeling and amplification product line, but equivalent products areavailable.

Array Hybridization and Processing (Whole Blood)

After pre-hybridization for 20 minutes at 45° C., 1.5 μg of each targetcDNA is mixed with Affymetrix hybridization controls in hybridizationbuffer and hybridized with the Feline-2 GeneChip® for 16 hours at 45° C.After the hybridization cocktails are removed, the chips are washed in afluidics station with low-stringency buffer (6× standard salinephosphate with EDTA, 0.01% Tween 20) and high stringency buffer (100 mMN-morpholino-ethanesulfonic acid (MES), 0.1 M NaCl, and 0.01% Tween 20)and stained with SAPE (streptavidin phycoerythrin). This process isfollowed by incubation with normal goat IgG and biotinylated mouseanti-streptavidin antibody (Vector Lab, BA-0500) followed by re-stainingwith SAPE. The chips are scanned in a GeneChip® Scanner 3000 7G(Affymetrix Inc, Santa Clara, Calif.) to detect hybridization signals.Image inspection is performed manually immediately following each scan(GeneChip® Expression Analysis Technical Manual. P/N 702232 Rev. 3,Chapter II and III).

Array Hybridization and Processing (Solid Tissues)

After pre-hybridization for 10 minutes at 45° C., 4.4 μg of each targetcDNA is mixed with Affymetrix hybridization controls in a hybridizationbuffer and hybridized with the Feline-2 GeneChip® for 16-18 hours at 45°C. After the hybridization cocktails are removed, the chips are washedin a fluidics station with low-stringency buffer (6× standard salinephosphate with EDTA, 0.01% Tween 20) and high stringency buffer (100 mMN-morpholino-ethanesulfonic acid (MES), 0.1 M NaCl, and 0.01% Tween 20)and stained with SAPE (streptavidin phycoerythrin). This process isfollowed by incubation with normal goat IgG and biotinylated mouseanti-streptavidin antibody (Vector Lab, BA-0500) followed by re-stainingwith SAPE. The chips are scanned in a GeneChip® Scanner 3000 7G(Affymetrix Inc, Santa Clara, Calif.) to detect hybridization signals.Image inspection is performed manually immediately following each scan(GeneChip® Expression Analysis Technical Manual. P/N 702232 Rev. 3,Chapter II and III).

Data Analysis

The Partek® GS (Partek Inc., St. Charles, Mo.) for Gene Expression Datasoftware (Partek Incorporated, 12747 Olive Blvd., Suite 205, St. Louis,Mo. 63141, U.S.A. http://www.partek.com/partekgs_geneexpression) is usedfor data analysis. The Robust Multichip Average (RMA) algorithm (Rafael.A. Irizarry, Benjamin M. Bolstad, Francois Collin, Leslie M. Cope,Bridget Hobbs and Terence P. Speed (2003), Summaries of AffymetrixGeneChip probe level data Nucleic Acids Research 31(4):e15) is used forbackground adjustment, normalization, and probe-level summarization ofthe GeneChip® samples. The ANOVA analysis is performed to findsignificant differentially expressed genes between any two groups with aminimal FDR control at 0.1 and a fold change of 1.25 in each direction.Our empirical studies have revealed that the Feline-2 GeneChips® have anassociated background noise level of 1.3 fold. Therefore, all analysespresented in this report employed a +/−1.25 fold cut-off (to be moreinclusive). Furthermore, the false discovery rate threshold of 0.1(means that 10% of observations are due to chance) is chosen as theminimum level of acceptable statistical significance.

This study focuses on gene expression in thyroid tissue and whole bloodwith respect to feline hyperthyroidism. In the tissue study, 1308 genesare found to be significantly differentially expressed betweenhyperthyroid felines and non-hyperthyroid felines with FDR=0.1 and foldchange>=+/−1.25. In the whole blood study, 1094 significant genes areidentified using the same cutoffs.

PCA analysis of both studies demonstrate that based on differential geneexpression, the hyperthyroid felines are clustered together and areseparate from the non-hyperthyroid felines.

Feline hyperthyroidism is an endocrine disorder that involves theproduction of too much thyroid hormone by the thyroid gland. Thiscondition tends to affect middle-aged and older felines, and both malesand females are equally at risk of developing this disorder.Hyperthyroidism affects every organ and cell in a feline's body (1).

The level of the thyroid hormone thyroxin (T4) is measured in the blood.High levels of this hormone are very indicative of hyperthyroidism. Inthe majority of felines suffering from this endocrine disorder, T4levels will be so high that there will be no question thathyperthyroidism is the problem. However, on some occasions a feline'shormone levels will fall within the upper levels of the normal range. Insuch cases, a further test known as a Free T4 (FT4) will be performed.In most cases, this second thyroid test is enough to confirm a diagnosisof hyperthyroidism.

Also, blood tests will reveal elevated levels of red blood cells andleukocytes along with low levels of lymphocytes and eosinophils. Also,some felines with hyperthyroidism will have elevated levels of ALT.Other substances may be present in elevated levels as well, such asother enzymes, the chemical creatinine, phosphorus, and the bile pigmentbilirubin. Some of these higher than normal levels are triggered byphysiological complications that arise from hyperthyroidism.

A feline's thyroid is a butterfly-shaped gland located in their neck.This gland is responsible for producing the hormone thyroxin (T4), whichplays a significant role in regulating a body's metabolic rate. Allorgans and physiological systems are affected by hyperthyroidism,causing classic symptoms such as weight loss, hyperactivity, increasedblood pressure, and an elevated heart rate.

In the tissue study, all of the genes involved in the pathway leading tothe production of thyroid hormones are found to be up-regulated in thehyperthyroid felines. The genes are those found in Table A. Theincreased expression of these genes contribute to a high T4 productionin feline body.

Description of Up-Regulated Genes that Contribute to Thyroid HormoneOver Production

IYD (4.55 fold increase) This gene encodes an enzyme that catalyzes theoxidative NADPH-dependent deiodination of mono- and diiodotyrosine,which are the halogenated byproducts of thyroid hormone production. TheN-terminus of the protein functions as a membrane anchor. Mutations inthis gene cause congenital hypothyroidism due to dyshormonogenesis type4, which is also referred to as deiodinase deficiency, or iodotyrosinedehalogenase deficiency, or thyroid hormonogenesis type 4.

TG (2.02 fold increase) Thyroglobulin (Tg) is a glycoprotein homodimerproduced predominantly by the thyroid gland. It acts as a substrate forthe synthesis of thyroxine and triiodothyronine as well as the storageof the inactive forms of thyroid hormone and iodine. Thyroglobulin issecreted from the endoplasmic reticulum to its site of iodination, andsubsequent thyroxine biosynthesis, in the follicular lumen. Mutations inthis gene cause thyroid dyshormonogenesis, manifested as goiter, and areassociated with moderate to severe congenital hypothyroidism.

SLC5A5, NIS (4.6 fold increase) This gene encodes a member of the sodiumglucose cotransporter family. The encoded protein is responsible for theuptake of iodine in tissues such as the thyroid and lactating breasttissue. The iodine taken up by the thyroid is incorporated into themetabolic regulators triiodothyronine (T3) and tetraiodothyronine (T4).Mutations in this gene are associated with thyroid dyshormonogenesis 1.

TPO (4.06 fold increase) This gene encodes a membrane-boundglycoprotein. The encoded protein acts as an enzyme and plays a centralrole in thyroid gland function. The protein functions in the iodinationof tyrosine residues in thyroglobulin and phenoxyester formation betweenpairs of iodinated tyrosines to generate the thyroid hormones, thyroxineand triiodothyronine. Mutations in this gene are associated with severaldisorders of thyroid hormonogenesis, including congenitalhypothyroidism, congenital goiter, and thyroid hormone organificationdefect IIA.

TSHR (2.2 fold increase) The protein encoded by this gene is a membraneprotein and a major controller of thyroid cell metabolism. The encodedprotein is a receptor for thyrothropin and thyrostimulin, and itsactivity is mediated by adenylate cyclase. Defects in this gene are acause of several types of hyperthyroidism.

DUOX2, ThOX (1.55 fold increase) The protein encoded by this gene is aglycoprotein and a member of the NADPH oxidase family. The synthesis ofthyroid hormone is catalyzed by a protein complex located at the apicalmembrane of thyroid follicular cells. This complex contains an iodidetransporter, thyroperoxidase, and a peroxide generating system thatincludes this encoded protein and DUOX1. This protein is known as dualoxidase because it has both a peroxidase homology domain and a gp91phoxdomain.

In hyperthyroid feline tissue study, several genes involved in thearachidonic acid production pathway are up regulated. Most of thosegenes are upstream genes as related to arachidonic acid synthesis,therefore, the production of arachidonic acid could be increased.Indeed, in human studies, it has been found that the arachidonic acidlevels are significantly higher in the hyperthyroid than either normalor in the hypothyroid group of people. Arachidonic acid is apolyunsaturated fatty acid that is present in the phospholipids(especially phosphatidylethanolamine, phosphatidylcholine andphosphatidylinositides) of membranes of the body's cells, and isabundant in the brain, muscles, liver. In addition to being involved incellular signaling as a lipid second messenger involved in theregulation of signaling enzymes, such as PLC-γ, PLC-δ and PKC-α, -β and-γ isoforms, arachidonic acid is a key inflammatory intermediate.Arachidonic acid plays a central role in inflammation related to injuryand many diseased states. How it is metabolized in the body dictates itsinflammatory or anti-inflammatory activity. Individuals suffering fromjoint pains or active inflammatory disease may find that increasedarachidonic acid consumption exacerbates symptoms, probably because itis being more readily converted to inflammatory compounds. Likewise,high arachidonic acid consumption is not advised for individuals with ahistory of inflammatory disease, or that are in compromised health. Itis also of note that while ARA supplementation does not appear to havepro-inflammatory effects in healthy individuals, it may counter theanti-inflammatory effects of omega-3 EFA supplementation. Therefore, inhyperthyroid felines, diet should be formatted to avoid arachidonicacid.

TABLE 2 Genes in Arachidonic Acid Production Pathway are Up-Regulated inthe Tissue Signal IDs HT/normal Gene Symbol Gene Name HP06563_at 4.36PA24A, cPLA2, phospholipase A2, PLA group IVA (cytosolic,calcium-dependent) HP16072_at; 2.4 ACSL5 acyl-CoA synthetase HP05605_atlong-chain family member 5 HP03050_at 1.62 PPT1 palmitoyl-proteinthioesterase 1 HP08203_at 1.46 LIPE lipase, endothelial HP07277_at 1.32ACSL3 acyl-CoA synthetase long-chain family member 3

In the whole blood gene expression study, 60 genes are found to overlapwith those found in the tissue analysis and exhibit the same directionof regulation. However, none of those genes found to be associated withthyroid hormone production as seen in the tissue study. By using these60 genes, the hyperthyroid felines can be distinguished from the normalones as tested in the blood, as well as in the tissue at the same time.The 60 genes are those found in Table B.

The invention claimed is:
 1. A method of treating a feline sufferingfrom hyperthyroidism, the method comprising: measuring an increase of atleast a three fold change in the level of expression of one or morebiomarkers in a sample from a feline to a control value for expressionin a sample from a normal feline or population of felines, and/orrelative to a previous individual baseline value from the feline;identifying the feline having an increase of at least three fold changein the level of expression as suffering from hyperthyroidism; andtreating the feline identified as suffering from hyperthryoidism bydietary intervention, wherein treating the feline by dietaryintervention comprises feeding the feline a diet comprising iodine in anamount of less than 0.35 mg/kg, selenium in an amount of less than 0.1mg/kg, and/or arachidonic acid in an amount of less than or equal to0.2%, and wherein the one or more biomarkers exhibiting the increase inthe level of expression comprises NIS.
 2. The method of claim 1, whereinmeasuring the increase of at least a three fold change in the level ofexpression of the one or more biomarkers comprises utilizing amicroarray or a polymerase chain reaction: wherein the microarray is aDNA microarray comprising one or more oligonucleotides complimentary tomRNA or cDNA corresponding to the one or more biomarkers to be measured,or wherein the polymerase chain reaction is a quantitative polymerasechain reaction with oligonucleotide primers for mRNA or cDNAcorresponding to the one or more biomarkers to be measured.
 3. Themethod of claim 1 wherein measuring the increase of at least a threefold change in the level of expression of the one or more biomarkerscomprises: isolating RNA from the sample, reverse transcribing the RNAto obtain the corresponding cDNA, isolating and fragmenting the cDNAthus obtained, contacting the cDNA fragments with a DNA microarraycomprising one or more oligonucleotides complementary to cDNAcorresponding to the one or more biomarkers to be measured, anddetecting hybridization between the cDNA fragments and the one or moreoligonucleotides in the DNA microarray.
 4. The method of claim 3,wherein the hybridization between the cDNA fragments and the one or moreoligonucleotides in the DNA microarray is under stringent conditions. 5.The method of claim 1, wherein the level of expression of the biomarkeris detected by an antibody to the expressed protein.
 6. The method ofclaim 1, wherein the level of expression of the biomarker is detected bymeasuring the amount of protein in the sample using quantitative massspectroscopy.
 7. The method of claim 1, wherein the level of expressionis measured for at least five biomarkers from the same sample.
 8. Themethod of claim 1, wherein the sample is blood or a thyroid tissue.
 9. Amethod of treating a feline suffering from hyperthyroidism, the methodcomprising: measuring an increase of at least a three fold change in thelevel of expression of one or more biomarkers in a sample from a felineto a control value for expression in a sample from a normal feline orpopulation of felines, and/or relative to a previous individual baselinevalue from the feline; and identifying the feline having an increase ofat least three fold change in the level of expression as suffering fromhyperthyroidism; and treating the feline identified as suffering fromhyperthyroidism by pharmaceutical intervention, wherein treating thefeline by pharmaceutical intervention comprises administering aneffective amount of a drug which inhibits production of T-4,administering radioiodine therapy, and/or performing a surgicalthyroidectomy, and wherein the one or more biomarkers exhibiting theincrease in the level of expression comprises NIS.
 10. The method ofclaim 9, wherein measuring the increase of at least a three fold changein the level of expression of the one or more biomarkers comprisesutilizing a microarray or a polymerase chain reaction: wherein themicroarray is a DNA microarray comprising one or more oligonucleotidescomplimentary to mRNA or cDNA corresponding to the one or morebiomarkers to be measured, or wherein the polymerase chain reaction is aquantitative polymerase chain reaction with oligonucleotide primers formRNA or cDNA corresponding to the one or more biomarkers to be measured.11. The method of claim 9, wherein measuring the increase of at least athree fold change in the level of expression of the one or morebiomarkers comprises: isolating RNA from the sample, reversetranscribing the RNA to obtain the corresponding cDNA, isolating andfragmenting the cDNA thus obtained, contacting the cDNA fragments with aDNA microarray comprising one or more oligonucleotides complementary tocDNA corresponding to the one or more biomarkers to be measured, anddetecting hybridization between the cDNA fragments and the one or moreoligonucleotides in the DNA microarray.
 12. The method of claim 9,wherein the level of expression of the biomarker is detected by anantibody to the expressed protein.
 13. The method of claim 9, whereinthe level of expression of the biomarker is detected by measuring theamount of protein in the sample using quantitative mass spectroscopy.14. The method of claim 9, wherein the sample is blood or thyroidtissue.
 15. A method of treating a feline suffering fromhyperthyroidism, the method comprising: measuring an increase of atleast a three fold change in the level of expression of one or morebiomarkers in a sample from a feline to a control value for expressionin a sample from a normal feline or population of felines, and/orrelative to a previous individual baseline value from the feline; andidentifying the feline having an increase of at least three fold changein the level of expression as suffering from hyperthyroidism; andtreating the feline identified as suffering from hyperthyroidism bypharmaceutical intervention, wherein treating the feline bypharmaceutical intervention comprises administering a drug selected frommethimazole or carbimazole, and wherein the one or more biomarkersexhibiting the increase in the level of expression comprises NIS. 16.The method of claim 15, wherein measuring the increase of at least athree fold change in the level of expression of the one or morebiomarkers comprises utilizing a microarray or a polymerase chainreaction: wherein the microarray is a DNA microarray comprising one ormore oligonucleotides complimentary to mRNA or cDNA corresponding to theone or more biomarkers to be measured, or wherein the polymerase chainreaction is a quantitative polymerase chain reaction witholigonucleotide primers for mRNA or cDNA corresponding to the one ormore biomarkers to be measured.
 17. The method of claim 15, whereinmeasuring the increase of at least a three fold change in the level ofexpression of the one or more biomarkers comprises: isolating RNA fromthe sample, reverse transcribing the RNA to obtain the correspondingcDNA, isolating and fragmenting the cDNA thus obtained, contacting thecDNA fragments with a DNA microarray comprising one or moreoligonucleotides complementary to cDNA corresponding to the one or morebiomarkers to be measured, and detecting hybridization between the cDNAfragments and the one or more oligonucleotides in the DNA microarray.18. The method of claim 15, wherein the level of expression of thebiomarker is detected by an antibody to the expressed protein.
 19. Themethod of claim 15, wherein the level of expression of the biomarker isdetected by measuring the amount of protein in the sample usingquantitative mass spectroscopy.
 20. The method of claim 15, wherein thesample is blood or thyroid tissue.